cDNA - (Jul/04/2009 )

Hi ev1,

Can anyone help me out? I'm having problem analysing my cDNA.

I used to run PCR straight after RT but i got no bands for result. so, my friend suggested i run a gel on the cDNA prod.i got nice band but i'm not sure whether it is the my cDNA prod. To normalise it, I tried running housekeeping genes of GAPDH, GST, cytp450 as positive control but all show no band. Thus, i cant prove tht the band was my expected cDNA. What should i do? I cant figure out what has gone wrong..

Has anyone try running cDNA on the gel? What ladder marker u guys used? Is it the cDNA expected size shld b same as the PCR prod. band size?

I upload my gel pic for u guys to help me analyse..thanks~ ^^

-7iew-

7iew on Jul 4 2009, 08:34 AM said:

Hi ev1,

Can anyone help me out? I'm having problem analysing my cDNA.

I used to run PCR straight after RT but i got no bands for result. so, my friend suggested i run a gel on the cDNA prod.i got nice band but i'm not sure whether it is the my cDNA prod. To normalise it, I tried running housekeeping genes of GAPDH, GST, cytp450 as positive control but all show no band. Thus, i cant prove tht the band was my expected cDNA. What should i do? I cant figure out what has gone wrong..

Has anyone try running cDNA on the gel? What ladder marker u guys used? Is it the cDNA expected size shld b same as the PCR prod. band size?

I upload my gel pic for u guys to help me analyse..thanks~ ^^

If you are talking about a specific cDNA product using gene-specific primers, your PCR product is you cDNA. Unless you are using primers that can give you full length cDNA, you can not assume full length cDNA size from your PCR product.

In any case, you should not see such smear. It should be a specific length band. And if there are splice-variants, more than one band. But not smear.

-cellcounter-

hi cellcounter,

hmm..i followed the standard protocol using oligo-dT primer to run RT..i guess tht wasnt gene specific is it? but i continued run PCR using a set of designed degenerate primer to get my specific seq of gene.

Any idea what way to reduce the smearing?

What i can do to correct my problem? I'm still very new in this RNA work..^^ Hope u can help me out..THanks a lot..

-7iew-

7iew on Jul 4 2009, 06:55 PM said:

hi cellcounter,

hmm..i followed the standard protocol using oligo-dT primer to run RT..i guess tht wasnt gene specific is it? but i continued run PCR using a set of designed degenerate primer to get my specific seq of gene.

Any idea what way to reduce the smearing?

What i can do to correct my problem? I'm still very new in this RNA work..^^ Hope u can help me out..THanks a lot..

You may see smears with degenerate primers, if they are not right sequences. These are not exactly gene-specific primers.

It depends upon how much degeneracy you have in the primers; you might just have to design more primers to cover all possible sequences, and even then, if there is some sequence variation, you may not be able to make it work.

Since you are new to this, I suggest you do some "working" RT-PCRs first (non-degenerate primers), if they work in your hands, the problem is not that of your technique.